Action of phospholipase A2 on phospholipid bilayers and biological membranes depends upon their organization. The experimental aim of this study is to characterize the factors governing such interactions in bilayers containing diacylphosphatidylcholine, lysophosphatidylcholine, and fatty acid. As a first step, binding of the enzyme to the bilayers will be studied in detail by fluorescence enhancement. The binding data is to be quantitated for the simplest equilibrium reaction E plus Ln less than greater than ELn, which represents the first presteady-state step in the catalytic cycle of the enzyme, and this step is followed by ELn plus S less than greater than Eln.S greater than ELn plus P. From binding isotherms we obtain the dissociation constant and N, the number of lipid molecules that bind to one enzyme molecule. The apparent dissociation constant (equalN.Kd) is expected to be related to the apparent kinetic Michaelis-Menten constant, Km. Thus we propose to measure the binding and kinetic parameters under comparable conditions to evaluate the effect of lipid composition and temperature. We will compare the binding and Kinetic parameters as a function of the structure of the lipid components (chain length, unsaturation, head group variation). This information will be correlated with the phase properties of the mixed lipid dispersions in order to evaluate the role of phase boundaries due to lateral phase separation as the possible phospholipase A2 binding sites. Such experiments are expected to provide basic information needed to elaborate the mechanism of interfacial activation of phospholipase A2. Activation of phospholipase A2 is thought to be the first and the rate limiting step in the biosynthesis of prostaglandins in vivo, since the action of phospholipase A2 releases arachidonic acid from appropriate phospholipids. All these studies will be conducted on phospholipase A2 from pig pancreas, and could be later extended to the enzyme from other sources.